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Sleep has been linked to the daily patterns of stress-responsive physiological systems, specifically the hypothalamic-pituitary-adrenal (HPA) axis and autonomic nervous system (ANS). However, extant research examining sleep and diurnal patterns of cortisol, the primary end product of the HPA axis, has primarily focused on sleep duration with limited attention on other facets of sleep. For example, it is not clear how specific aspects of sleep (e.g., sleep quality, sleep duration variability) are related to specific components of diurnal cortisol rhythms. Salivary alpha-amylase (sAA) has been recognized as a surrogate marker of ANS activity, but limited research has explored relations between sleep and sAA diurnal rhythms. The current study utilized an ecological momentary assessment protocol to examine within- and between-person relations between several facets of sleep behavior using multiple methods (e.g., subjective report, actigraphy) and salivary cortisol and sAA. Older adolescents (N=76) provided saliva samples and diary entries five times per day over the course of three days. Sleep was assessed via questionnaire, through daily diaries, and monitored objectively using actigraphy over a four day period. Between-person results revealed that shorter average objective sleep duration and greater sleep duration variability were related to lower levels of waking cortisol and flatter diurnal slopes across the day. Within-person results revealed that on nights when individuals slept for shorter durations than usual they also had lower levels of waking cortisol the next day. Sleep was not related to the cortisol awakening response (CAR) or diurnal patterns of sAA, in either between-person or within-person analyses. However, typical sleep behaviors measured via questionnaire were related to waking levels of sAA. Overall, this study provides a greater understanding of how multiple components of sleep, measured in naturalistic environments, are related to cortisol and sAA diurnal rhythms, and how day-to-day, within-person changes in sleep duration contribute to daily variations in cortisol., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

It is suggested that salivary alpha-amylase (sAA) may be a marker of sympathoadrenal medullary system activity. Thus, it can be a possible relationship sAA and anxiety disorders. The aim of this study is to investigate sAA in children and adolescents with anxiety disorders and healthy controls. Thirty drug-free youths, aged 8-16 years, who were diagnosed as any anxiety disorders and 36 healthy controls with similar socio-demographic characteristics were included in this study. The sAA was found to be significantly increased in anxiety group compared to control group. However, there was no correlation between sAA and any anxiety scores of the scales. Present study suggested that anxiety disorders in youths may be associated with increased autonomic activity., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Adenine and uracil nucleotides play key functions in the autonomic nervous system (ANS). For instance, ATP acts as a neurotransmitter, co-transmitter and neuromodulator in the ANS. The purinergic system encompasses (1) receptors that respond to extracellular purines, which are designated as P1 and P2 purinoceptors, (2) purine release and uptake, and (3) a cascade of enzymes that regulate the concentration of purines near the cell surface. Ectonucleotidases and adenosine deaminase (ADA) are enzymes responsible for the hydrolysis of ATP (and other nucleotides such as ADP, UTP, UDP, AMP) and adenosine, respectively. Accordingly, these enzymes are expected to play an important role in the control of neuro-effector transmission in tissues innervated by both the sympathetic and parasympathetic divisions of the ANS. Indeed, ectonucleotidases have the ability to either terminate P2 receptor responses initiated by nucleoside triphosphates (ATP and UTP), and/or to favor the activation of ADP (e.g. P2Y1,12,13) and UDP (e.g. P2Y6) and/or adenosine (P1) specific receptors. In addition, ectonucleotidases can also importantly protect some P2 receptors from desensitization (e.g. P2X1, P2Y1). In this review, we present the (putative) roles of ectonucleotidases and ADA in the ANS with a focus on their regulatory activity at neuro-effector junctions in the following tissues: heart, vas deferens, urinary bladder, salivary glands, blood vessels and the intestine. We also present their implication in nociceptive transmission., (Copyright © 2015. Published by Elsevier B.V.)

Objective: Recently, salivary alpha-amylase (sAA) has been proposed as a suitable index for sympathetic activity and dysregulation of the autonomic nervous system (ANS). Although determinants of sAA have been described, they have not been studied within the same study with a large sample size without potential disturbances of psychopathology. In this paper, we report about correlates of evening sAA in saliva., Methods: In 487 participants (mean age=42.9years, 59.8% female) without lifetime psychiatric disorders from the Netherlands Study of Depression and Anxiety (NESDA), sociodemographic, health and sampling determinants of sAA levels were examined using multivariable linear regression analysis. sAA was measured in two saliva samples that participants collected in the late evening, at 22:00h and 23:00h, after which these were averaged., Results: In multivariate analysis, age (β=0.20, p<0.001) and daily alcohol intake (β=-0.13, p=0.01) were independent determinants of evening sAA levels. Gender, allergy or lung disease, and the use of oral contraceptives were univariate correlates, but no longer associated with sAA in the multivariate model., Conclusions: Age and alcohol use were identified as potential confounding factors that should be taken into account in epidemiologic studies that examine the ANS function using sAA., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Mineralocorticoid excess increases superoxide production by activating NADPH oxidase (NOX), and intracerebroventricular infusions of NADPH oxidase inhibitors attenuate aldosterone (Aldo)/salt-induced hypertension. It has been hypothesized that increased reactive oxygen species (ROS) in the brain may be a key mechanism in the development of hypertension. The present study investigated the brain regional specificity of NADPH oxidase and the role of NOX2 and NOX4 NADPH oxidase subunits in the hypothalamic paraventricular nucleus (PVN) in Aldo/salt-induced hypertension. PVN injections of adenoviral vectors expressing small interfering (si)RNA targeting NOX2 (AdsiRNA-NOX2) or NOX4 (AdsiRNA-NOX4) mRNAs were used to knock down NOX2 and NOX4 proteins. Three days later, delivery of Aldo (0.2 mg·kg(-1)·day(-1) sc) via osmotic pump commenced and 1% NaCl was provided in place of water. PVN injections of either AdsiRNA-NOX2 or AdsiRNA-NOX4 significantly attenuated the development of Aldo/NaCl-induced hypertension. In an additional study, Aldo/salt-induced hypertension was also significantly attenuated in NOX2 (genomic) knockout mice compared with wild-type controls. When animals from both functional studies underwent ganglionic blockade, there was a reduced fall in blood pressure in the NOX2 and NOX4 knockdown/knockout mice. Western blot analyses of the PVN of siRNA-NOX2- or siRNA-NOX4-injected mice confirmed a marked reduction in the expression of NOX2 or NOX4 protein. In cultured PVN neurons, silencing either NOX2 or NOX4 protein production by culturing PVN cells with siRNA-NOX2 or siRNA-NOX4 attenuated Aldo-induced ROS. These data indicate that both NOX2 and NOX4 in the PVN contribute to elevated sympathetic activity and the hypertensivogenic actions induced by mineralocorticoid excess.

Tetrahydrobiopterin (BH4) functions as a cofactor in several important enzyme systems. Substantial evidence implicates BH4 as a key regulator of endothelial nitric oxide synthase (eNOS) in the setting of endothelial dysfunction and atherosclerosis. Investigators have now taken early steps in addressing the potential of BH4 as a therapeutic strategy. However, it has become more apparent that the role of BH4 in other enzymatic pathways, including other NOS isoforms and the aromatic amino acid hydroxylases, may have a bearing on important aspects of cardiovascular homeostasis. Together with eNOS, these enzymes may play key roles in diverse cardiovascular disease states such as ischaemia-reperfusion injury, cardiac hypertrophy, cardiac autonomic function and pulmonary hypertension. This review provides an overview of the role of BH4 in cardiovascular pathophysiology.

The catecholamine biosynthetic pathway consists of several enzymatic steps in series, beginning with the amino acids phenylalanine and tyrosine, and eventuating in the catecholamines norepinephrine (noradrenaline) and epinephrine (adrenaline). Since the enzyme tyrosine hydroxylase (TH; tyrosine 3-mono-oxygenase; EC 1.14.16.2; chromosome 11p15.5) is generally considered to be rate-limiting in this pathway, probed as to whether common genetic variation at the TH gene occurred, and whether such variants contributed to inter-individual alterations in autonomic function, either biochemical or physiological. We began with sequencing a tetranucleotide (TCAT) repeat in the first intron, and found that the two most common versions, (TCAT)(6) and (TCAT)(10i), predicted heritable autonomic traits in twin pairs. We then conducted systematic polymorphism discovery across the ~8 kbp locus, and discovered numerous variants, principally non-coding. The proximal promoter block contained four common variants, and its haplotypes and SNPs (especially C-824T, rs10770141) predicted catecholamine secretion, environmental stress-induced BP increments, and hypertension. Finally, we found that two of the common promoter variants, C-824T (rs10770141) and A-581G (rs10770140), were functional in that they differentially affected transcriptional activity of the isolated promoter, disrupted recognition motifs for specific transcription factor binding, altered the promoter responses to the co-transfected (exogenous) factors, and bound the endogenous factors in the chromatin fraction of the nucleus. We concluded that common variation in the proximal TH promoter is functional, giving rise to changes in autonomic function and consequently cardiovascular risk.

Angiotensin-converting enzyme 2 (ACE2) is a new component of the renin-angiotensin system (RAS). Accumulating evidence shows that ACE2 provides protective effects in peripheral tissues and has great potential for the treatment of RAS-related diseases. The role of ACE2 in the central nervous system is not well established. However, in recent years, much more progress has been made on the studies of this carboxypeptidase in the central regulation of blood pressure and cardiovascular function in general. It has been shown that brain ACE2 interacts with the other components of the RAS (ACE, angiotensin II, and angiotensin II type 1 receptor), protects baroreflex and autonomic function, stimulates nitric oxide release, reduces oxidative stress, and prevents the development of or attenuates hypertension. These data support the critical role of ACE2 in the central regulation of cardiovascular function. This review summarizes recently published data on the central effects of ACE2 in the regulation of cardiovascular function.

Chronic myocardial infarction (CMI) is associated with remodeling of the ventricle and evokes adaption in the cardiac neurohumoral control systems. To evaluate the remodeling of the intrinsic cardiac nervous system following myocardial infarction, the dorsal descending coronary artery was ligated in the guinea pig heart and the animals were allowed to recover for 7-9 wk. Thereafter, atrial neurons of the intrinsic cardiac plexus were isolated for electrophysiological and immunohistochemical analyses. Intracellular voltage recordings from intrinsic cardiac neurons demonstrated no significant changes in passive membrane properties or action potential configuration compared with age-matched controls and sham-operated animals. The intrinsic cardiac neurons from chronic infarcted hearts did demonstrate an increase in evoked action potential (AP) frequency (as determined by the number of APs produced with depolarizing stimuli) and an increase in responses to exogenously applied histamine compared with sham and age-matched controls. Conversely, pituitary adenylate cyclase-activating polypeptide (PACAP)-induced increases in intrinsic cardiac neuron-evoked AP frequency were similar between control and CMI animals. Immunohistochemical analysis demonstrated a threefold increase in percentage of neurons immunoreactive for neuronal nitric oxide synthase (NOS) in CMI animals compared with control and the additional expression of inducible NOS by some neurons, which was not evident in control animals. Finally, the density of mast cells within the intrinsic cardiac plexus was increased threefold in preparations from CMI animals. These results indicate that CMI induces a differential remodeling of intrinsic cardiac neurons and functional upregulation of neuronal responsiveness to specific neuromodulators.

Dopamine beta-hydroxylase (DbetaH) deficiency is a very rare form of primary autonomic failure characterized by a complete absence of noradrenaline and adrenaline in plasma together with increased dopamine plasma levels. The prevalence of DbetaH deficiency is unknown. Only a limited number of cases with this disease have been reported. DbetaH deficiency is mainly characterized by cardiovascular disorders and severe orthostatic hypotension. First symptoms often start during a complicated perinatal period with hypotension, muscle hypotonia, hypothermia and hypoglycemia. Children with DbetaH deficiency exhibit reduced ability to exercise because of blood pressure inadaptation with exertion and syncope. Symptoms usually worsen progressively during late adolescence and early adulthood with severe orthostatic hypotension, eyelid ptosis, nasal stuffiness and sexual disorders. Limitation in standing tolerance, limited ability to exercise and traumatic morbidity related to falls and syncope may represent later evolution. The syndrome is caused by heterogeneous molecular alterations of the DBH gene and is inherited in an autosomal recessive manner. Restoration of plasma noradrenaline to the normal range can be achieved by therapy with the synthetic precursor of noradrenaline, L-threo-dihydroxyphenylserine (DOPS). Oral administration of 100 to 500 mg DOPS, twice or three times daily, increases blood pressure and reverses the orthostatic intolerance.

Nitric oxide (NO) is a potent modulator of cardiac and vascular regulation. Its role in cardiac-autonomic neural signaling has received much attention over the last decade because of the ability of NO to alter cardiac sympathovagal balance to favor more anti-arrhythmic states. Complexity and controversy have arisen, however, because of the numerous sources of NO in the brain, peripheral nerves, and cardiomyocytes, all of which are potential regulators of cardiac excitability and calcium signaling. This review addresses the integrative role of NO as a relatively ubiquitous signaling molecule with respect to cardiac neurobiology. The present idea, that divergent NO-signaling pathways from multiple sources within the heart and nervous system converge to modulate cardiac excitability and impact on morbidity and mortality in health and disease, is discussed.

Tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, has a common tetranucleotide repeat polymorphism, (TCAT)(n). We asked whether variation at (TCAT)(n) may influence the autonomic nervous system and its response to environmental stress. To understand the role of heredity in such traits, we turned to a human twin study design. Both biochemical and physiological autonomic traits displayed substantial heritability (h(2)), up to h(2) = 56.8 +/- 7.5% (P < 0.0001) for norepinephrine secretion, and h(2) = 61 +/- 6% (P < 0.001) for heart rate. Common (TCAT)(n) alleles, particularly (TCAT)(6) and (TCAT)(10i), predicted such traits (including catecholamine secretion, as well as basal and poststress heart rate) in allele copy number dose-dependent fashion, although in directionally opposite ways, indicating functional allelic heterogeneity. (TCAT)(n) diploid genotypes (e.g., [TCAT](6)/[TCAT](10i)) predicted the same physiological traits but with increased explanatory power for trait variation (in contrast to allele copy number). Multivariate ANOVA documented genetic pleiotropy: joint effects of the (TCAT)(10i) allele on both biochemical (norepinephrine) and physiological (heart rate) traits. (TCAT)(6) allele frequencies were lower in normotensive twins at genetic risk of hypertension, consistent with an effect to protect against later development of hypertension, and suggesting that the traits predicted by these variants in still-normotensive subjects are early, heritable, "intermediate phenotypes" in the pathogenetic scheme for later development of sustained hypertension. We conclude that common allelic variation within the tyrosine hydroxylase locus exerts a powerful, heritable effect on autonomic control of the circulation and that such variation may have implications in later development of cardiovascular disease traits such as hypertension.

The innervation of the cervine testis was studied in 6 roe deers, 7 red deers and 14 fallow deers. The results for the three species are rather similar. With anti-sera to neurofilament (NF) and neuron specific enolase (NSE), all small and large nerve fascicles can be demonstrated, but single fibers are incompletely stained. Immunoreactions against protein gene product-9.5 (PGP-9.5) and GAP-43 (growth-associated protein-43) are better suited to depict the complete innervation pattern. Bundles of the superior spermatic and inferior spermatic nerves reach the testis via three access routes as funicular, mesorchial and caudal nerve contributions. We found no morphological evidence that the nerves in the cervine testis are directly involved in regulating Leydig cell function or seminiferous tubular motility. The majority of the testicular nerves are associated with the testicular arteries, but the musculature in the walls of the venous plexus pampiniformis is also innervated. All vascular nerve fibers represent postjunctional sympathetic axons displaying a strong dopamine-beta-hydroxylase (DBH) activity, mostly co-expressed with neuropeptide Y (NPY). The presence of cholinergic fibers in the testis of the deer is only sporadic and probably of no functional importance. In all three species of deer, a small quantity of myelinated nerve fibers is encountered in spermatic cord and tunica albuginea and regarded as afferent. The viscerosensory quality in the testicular intrinsic innervation is very likely mediated by the CGRP (calcitonin gene-related peptide)-positive fibers that run independently from the testicular vessels and end in the connective tissue of spermatic cord and tunica albuginea. The testis of the red deer contains significantly more VIP (vasoactive intestinal polypeptide)-positive axons than that of roe and fallow deer. The nerve density in the interior of the testicular lobules shows no regional differences, but there are age- and season-related changes that correlate with the developmental and functional state of the seminiferous tubules. Small testes with solid and narrow tubules, as seen in the prepuberal phase and during seasonal reproductive quiescence, are better innervated than large testes with expanded and spermatogenetically active seminiferous tubules.

In the present work, histochemical and biochemical studies were conducted to analyze changes in the pattern of autonomic innervation during sexual maturation, using the rat epididymis as a model. Glyoxylic acid histochemistry and immunohistochemical studies against dopamine beta-hydroxylase (DbetaH) and acetylcholinesterase (AChE) indicated a reduction in the amount of catecholaminergic and AChE-positive neurons, fibers, and puncta detected in the cauda epididymis of adult rats (120 days old), when compared to immature (40 days) and young adult (60 days) animals. No obvious age-related variations were detected in the few catecholaminergic and AChE-positive fibers and puncta present in the caput region. AChE-positive fibers were found sorting out among epithelial cells and ending free upon the epithelial surface or into the tubular lumen of the cauda region of adult rats. Furthermore, a positive staining for AChE in epithelial cells was also detected in the caput and cauda epididymis in all ages studied. Biochemical analysis confirmed a significant decrease in noradrenaline concentration as well as AChE activity in the cauda epididymis with sexual maturation. Immunohistochemical studies against microtubule-associated protein 1B (MAP 1B), a neuronal cytoskeletal marker, further substantiated the quantitative changes observed in catecholaminergic and AChE-positive neuronal elements in the cauda epididymis. Thus, our results documented segment-specific variations in noradrenaline concentration and AChE activity during epididymal sexual maturation and suggest that such variations result, at least in part, from the refinement of the autonomic innervation pattern with age.

Our laboratory has generated a genetically mutant mouse in which the alpha subunit of the heterotrimeric GTP binding protein, G(z) has been made dysfunctional by homologous recombination to determine its in vivo function. These animals show a characteristic failure to thrive phenotype. G(z alpha) is expressed in a variety of nervous system tissues as well as in the adrenal medulla. We therefore examined the autonomic nervous system of the G(z alpha) deficient mouse by measuring the activity of tyrosine hydroxylase and choline acetyltransferase in the superior cervical ganglia, submaxillary gland and the adrenal medulla. Preliminary results using animals of mixed BALB/c and C57BL/6 strains gave inconsistent results. Further experiments demonstrated differences in the activity of tyrosine hydroxylase and choline acetyltransferase between BALB/c and C57BL/6 mouse strains. The analysis of the pure strains showed a reduction in the size and enzyme levels of the adrenal gland and submaxillary glands of the G(z alpha) deficient mouse suggesting a role for adrenal insufficiency and/or nutritional disorders for the failure to thrive phenotype. The survival of sympathetic and sensory neurons was also examined in the G(z alpha) deficient mouse and in the presence of pertussis toxin, sympathetic but not sensory neuronal survival in G(z alpha) deficient mice was significantly attenuated. This suggests that in vivo other pertussis toxin sensitive G proteins may be recruited to compensate for the loss of G(z alpha).

Purpose: To determine the expression patterns of the vesicular acetylcholine transporter (VAChT), tyrosine hydroxylase (TH) and neuronal nitric oxide synthase (nNOS) in the pterygopalatine ganglion (PPG) and the exorbital lacrimal gland of normal mice., Methods: Mouse PPG and lacrimal glands were processed for single- and double-labeled indirect immunofluorescence studies. Slides were examined with conventional fluorescence microscopy and confocal laser scanning microscopy., Results: All the somata in the PPG expressed both VAChT and nNOS immunoreactivity (IR). The postganglionic axons within the ganglion showed less VAChT-immunoreactive intensity than that seen in the somata, whereas nNOS IR was almost undetectable. In the lacrimal gland, nNOS-positive nerve bundles and fibers were observed to be associated with tear-collecting ducts, blood vessels, and acini. Some nNOS-positive punctate elements appeared to be distributed among acini. Many nerve fibers were VAChT immunoreactive and a small number of fibers were TH immunoreactive in the gland. Most of the VAChT-positive fibers and some of the TH-positive nerves displayed nNOS IR., Conclusions: The expression of nNOS in cells of the PPG and in lacrimal gland nerves suggests that NO may play a role in modulating tear production. The site of action may include the PPG, ducts, blood vessels, acini, nerve fibers, and myoepithelial cells within the gland. NO may modulate parasympathetic and/or sympathetic synaptic transmission or by acting directly on lacrimal gland components. The interaction between NO-ergic and the conventional autonomic input illustrates the complexity of the innervation pattern of the mouse lacrimal gland.

ensp;The distribution and colocalisation of nicotinamide adenine dinucleotide phosphate reduced-diaphorase (NADPH-d)-/nitric oxide synthase (NOS)-containing (nitrergic) neurons in the innervation of the duck ureter have been studied using histochemistry and immunohistochemistry. Quantitative analysis showed that nitrergic neurons made up 60% and 70% of the total intramural and adventitial neuronal populations, respectively. About 40% of intramural nitrergic neurons expressed VIP-immunoreactivity, and about 75% of nitrergic adventitial neurons expressed TH-immunoreactivity. The density of nitrergic adventitial neurons was significantly greater in the lower tract than in the upper and intermediate tracts. Nerve lesioning experiments showed that the majority of ureteral nitrergic innervation was extrinsic in origin; nitrergic adventitial neurons primarily projected caudocranially, whereas NOS-immunoreactive and NOS-/VIP-immunoreactive intramural neurons primarily projected craniocaudally. These findings suggest that, in birds, the nitrergic innervation plays a role in ureteral functions such as epithelial mucosecretion, muscular motility, and the closing and/or opening of the ureteral papilla.

Purpose: The aim of present study was to determine the topographic relationship between heme oxygenase-2 (HO-2), which synthesizes carbon monoxide (CO), and neuronal nitric oxide synthase (nNOS), which generates nitric oxide (NO), in the autonomic nerves of the human ductus deferens and seminal vesicle., Materials and Methods: Specimens of the ductus deferens and seminal vesicle were obtained during cancer surgery or vasectomy. HO-2 and nNOS were localized by indirect immunofluorescence. Additionally, the histochemical NADPH-diaphorase (NADPH-d) activity of NOS was demonstrated using a standard staining method and some modifications., Results: Anti-HO-2 labeling stained virtually all nerve cell bodies in local ganglia of the pelvic plexus, which is composed of a mixed population of postganglionic sympathetic and parasympathetic neurons supplying the pelvic viscera. Furthermore, nerve cell bodies in the wall of the seminal vesicle, which are considered an extension of the pelvic plexus, were also found to stain positively for HO-2. Some of the HO-2-immunoreactive ganglion cells were also nNOS-positive, their proportion varying between individual ganglia but generally not exceeding 20%. Both enzymes were present in large adventitial nerve trunks. Only nNOS but no HO-2 was found in small intramuscular and mucosal nerve fibers. In both the ductus deferens and seminal vesicle, the highest density of nNOS-containing nerve fibers was in the lamina propria of the mucosa. A well-developed plexus of nNOS-positive nerve fibers was also observed in the muscular layer of the seminal vesicle. By contrast, there was a very sparse innervation by nNOS-positive nerve fibers in the muscle coat of the ductus deferens. In addition, a population of epithelial cells in the seminal vesicle may contain an isoform of NOS, as revealed by a resistant NADPH-d activity., Conclusions: These findings set the scene for functional studies which will hopefully clarify the biological role of CO and NO in the control of the ductus deferens and seminal vesicle.

Immunohistochemical, biochemical and functional studies have revealed two separate cholinergic systems in the arterial vascular wall. Endothelial cells represent the ubiquitous intrinsic, intimal system; they contain the acetylcholine-synthesizing enzyme, choline acetyltransferase, release a choline ester, and contain functional muscarinic receptors. Perivascular autonomic nerve fibres represent the extrinsic, adventitial system. These axons are not ubiquitous but show a highly selective distribution among and even within organs, and utilize co-mediators (NO, neuropeptides) in an organ-specific pattern. We put forward the hypothesis that the intrinsic, intimal system serves as a general regulator of basal vascular tone and wall structure responding to local, luminal stimuli, whereas the perivascular nerve fibres act on top of this basal tone by providing fine tuning in response to reflex activation due to systemic demands.

Previous studies used nicotinamide adenine diphosphate (NADPH)-diaphorase histochemistry as an indicator of nitric oxide synthase (NOS) expression in the adult mammalian cochlea. In this study, we investigated the early postnatal expression of diaphorase activity in the hamster cochlea. Two types of extrinsic fibers were intensely labeled as early as postnatal day 3 (P3) in the portion of the cochlear nerve that innervates the base of the modiolus. By P10, these fibers had reached the spiral ganglion and were projecting toward the organ of Corti. The perivascular type of fiber did not project into the organ of Corti; however, the nonperivascular type could be traced among the supporting cells below the outer hair cells. Spiral ganglion cell somata were also labeled as early as P3. The onset of diaphorase expression in the spiral ganglion cells corresponds to a critical period of synaptogenesis for these sensorineural cells. If NADPH-diaphorase activity is an indicator of NOS, then our results suggest that NO may play a role during postnatal cochlear development., (Copyright 1999 Wiley-Liss, Inc.)

It is known that the vagus nerve contains catecholaminergic fibers. However, the origin of these fibers has not been systematically examined. In this study, we addressed this issue using retrograde tracing from the subdiaphragmatic vagus nerve combined with immunocytochemistry. The cervical and thoracic sympathetic trunk ganglia, the nodose ganglia and the dorsal motor nucleus of the vagus nerve were examined following injection of Fluoro-Gold or cholera toxin horseradish peroxidase conjugate into the trunks of the subdiaphragmatic vagus nerve of rats. Numerous retrogradely labeled neurons were seen in the nodose ganglion and the dorsal motor nucleus of the vagus nerve. Very few labeled neurons were found in the sympathetic ganglia (less than 0.06% of the neurons in either superior cervical ganglion or cervicothoracic ganglion were retrogradely labeled). Double labeling with immunofluoresence for catecholamine synthesizing enzymes revealed that: (1) 92% of all Fluoro-Gold retrogradely labeled tyrosine hydroxylase immunoreactive neurons were found in parasympathetic sources (75% in the dorsal motor nucleus of the vagus nerve and 17% in the nodose ganglia), and only 8% in the cervicothoracic sympathetic ganglia; (2) 12% of the retrogradely labeled catecholaminergic neurons in the dorsal motor nucleus of the vagus nerve were also dopamine-beta-hydroxylase immunopositive neurons; (3) 70% of the retrogradely labeled neurons in the sympathetic ganglia were tyrosine hydroxylase immunopositive and 54% of these catecholaminergic neurons contained dopamine-beta-hydroxylase, while 30% of the retrogradely labeled neurons were non-catecholaminergic neurons. These results indicate that catecholaminergic fibers in the abdominal vagus nerve are primarily dopaminergic and of parasympathetic origin, and that only an extremely small number of these fibers, mostly noradrenergic in nature, arise from postganglionic sympathetic neurons.

Nitric oxide (NO) is a unique biological messenger molecule. It serves, in part, as a neurotransmitter in the central and peripheral nervous systems. Neurons containing NO have been identified histochemically by the presence of nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) reactivity or immunohistochemically by the antibody for neuronal NO synthase (n-NOS). Previous histochemical or pharmacological studies have raised the possibility that NO may play an important role in the neural pathways of the lower urinary tract. There is also considerable evidence to suggest that n-NOS is plastic and could be upregulated following certain lesions in the lower urinary tract. The present review summarises the distribution of n-NOS containing neurons innervating the urinary bladder and the changes of the enzyme expression in some experimentally induced pathological conditions.

Heart autonomic innervation was studied in dogs during the acute phase of the experimental infection with the Berenice-78 strain of Trypanosoma cruzi. A glyoxylic acid-induced fluorescence method for catecholamines and a thiocholine method for demonstrating acetylcholinesterase activity showed the sympathetic and the parasympathetic nerve fibers, respectively. At day 34 of infection, moderate-to-intense rarefaction of both cholinergic and noradrenergic nerve fibers occurred in the atria of all animals coincident with moderate to intense myocarditis. In the ventricles, sympathetic denervation was clearly present only when the inflammatory processes were moderate to intense. Preliminary results on the chronic phase indicate that normal autonomic innervation coexists with an incipient chronic fibrosing myocarditis.

The aim of this study was to evaluate the regulatory role of vagal afferents in the development of colonic inflammation induced by trinitrobenzenesulfonic acid (TNBS) in rats. Groups of Wistar rats were treated with capsaicin or its vehicle applied perivagally (sham treatment). Colonic transit time was evaluated, and, two days later, one half of the animals received an intracolonic instillation of TNBS/ethanol (40 mg/kg), and the other received saline. Inflammation was evaluated functionally (gut permeability), biochemically (myeloperoxydase activity) and histologically. Vagal capsaicin deafferentation did not modify colonic transit time. In TNBS treated groups, inflammation was enhanced by capsaicin pretreatment, as determined by an increased gut permeability, MPO activity, and histological damage score. These results suggest that vagal afferents have a protective role in TNBS-induced colitis in rats, unrelated to changes in colonic transit time.

The structure of the nervous network and the distribution of tyrosine hydroxylase (TH)- and various neuropeptide-containing nerves were immunohistochemically studied in the glottis of the dog. The nervous network in the glottis revealed apparent regional differences in morphology. The nervous network in the cartilaginous vocal fold of the posterior glottis consisted of nerve bundles running parallel to the edge of the vocal fold. Only a small number of nerve bundles were observed in the anterior glottis, specifically in membranous vocal fold. In the subepithelial layer of the posterior glottis, a moderate number of galanin (GAL)-immunoreactive nerve fibers were observed, while only a few fibers were present in the anterior glottis. Numerous vasoactive intestinal peptide (VIP)-, GAL-, methionine-enkephalin (ENK)- and TH-immunoreactive nerve fibers were observed within and around the laryngeal submucosal seromucous gland. Many TH- and neuropeptide Y (NPY)-immunoreactive fibers were arranged around the blood vessels. In the epithelia, free nerve endings with immunoreactivity for substance P (SP) and calcitonin gene-related peptide (CGRP) was observed. Furthermore, nerve cell bodies with SP-, VIP-, GAL-, ENK-, and NPY-immunoreactivity were observed in the deep region of the submucosal layer. The results from the present study suggest that there is autonomic regulation of the glottis. Regional structural differences in the nervous network of the glottis may reflect functional differences.

1. Nitric oxide (NO) is formed by neuronal NO synthase (nNOS) and acts as a non-conventional neurotransmitter in the brain. A growing body of evidence supports the hypothesis that NO acts to decrease sympathetic output to the periphery; these effects may occur at several autonomic sites. The present review describes studies from our laboratory that address this hypothesis. 2. Restraint stress activates putative NO-producing neurons in many autonomic centres: preoptic area, medial septum, amygdala, hypothalamus, including the paraventricular nucleus (PVN), raphe nuclei, nucleus tractus solitarius (NTS) and ventrolateral medulla (VLM). These results suggest that NO is directly or indirectly involved in regulating sympathetic output to the periphery. 3. Systemic angiotensin II (AngII) activates putative NO-producing neurons in the PVN. These neurons may be activated either by the increases in arterial pressure that accompany AngII injections or due to activation of AngII-containing neural pathways. 4. Hypotension is associated with the activation of putative NO-producing PVN neurons, small numbers of which also project to the NTS or VLM. As the majority of activated neurons is in the magnocellular division, NO production may be related to the production of vasopressin. 5. Adult spontaneously hypertensive rats (SHR) show increased gene expression of nNOS in the hypothalamus, dorsal medulla and caudal VLM. These differences are not present in young prehypertensive SHR, suggesting that the changes in gene expression in adult rats are associated with the increased sympathetic nerve activity found in these rats. 6. Gene expression of nNOS is altered in the hypothalamus and caudal VLM of renal hypertensive rats at 3 and 6 weeks after surgical induction of hypertension. Contrasting results at the two time points may be due to differing underlying physiological processes that characterize the two stages of renal hypertension. 7. Nitric oxide may affect sympathetic output through several possible mechanisms. These include affecting production of the second messenger cGMP and interactions with more classical neurotransmitters or with neurohormonal systems in the brain.

The innervation of the porcine testis was studied in 20 pigs, aged from 3 days to 2.5 years, and revealed remarkable changes in the period from birth to adulthood. Testes in piglets of 3 to 5 weeks have the most intense and most constant innervation, which reaches the gonad by three different routes: the funicular, caudal and mesorchial. Nerve fibers supply the vascular structures of the spermatic cord, the tunica albuginea, nearly all the septula testis and the mediastinum. Only exceptionally are axons in contact with Leydig cells. Nearly all the testicular nerves are positive for DBH and therefore represent postganglionic sympathetic axons. From their association with blood vessels it can be concluded that the majority of nerves are vasomotor in function. No cholinergic and myelinated fibers can be detected in the porcine testis. NPY-immunoreactive fibers are the dominating peptide-containing neuronal component. In the testes of 3- and 7-day-old piglets the degree of septal and mediastinal innervation is significantly smaller than in 3- to 5-week-old animals. In 7- to 10-week-old pigs, testicular innervation shows varying degrees of withdrawal, and the testes of adult boars are completely devoid of intrinsic nerves. Only the funicular nerves supplying the testicular artery and pampiniform plexus are preserved in the adult age group. So, the vasomotor control of intratunical, septal and mediastinal vessels and of the complete micro-circulation within the testicular parenchyma is effected without any direct nerve participation in the sexually mature boar.

A missense mutation (Gly93-->Ser) was identified in exon 4 of the Cu/Zn superoxide dismutase (SOD1) gene of a 48-year-old Japanese man with familial amyotrophic lateral sclerosis (FALS). The SOD1 activities in the fibroblasts and in a lysate of erythrocytes of the patient did not differ from those of healthy controls. The clinical characteristics of the patient were fairly slow progression of the illness, prominent sensory impairment, urinary disturbance and blood pressure fluctuation due to sympathetic hyperactivity. The severe sensory and autonomic disturbances, association of which with FALS has not been previously reported, may represent specific clinical features associated with the Gly93Ser mutation or may indicate the variability of clinical findings even in patients with the same mutation.

The neurones and microvessels of the dorsal motor nucleus of the vagus (DMN), the nucleus ambiguus (NA) and the nucleus tractus solitarius (NTS) of 4, 24 and 30 month male Wistar rats have been examined morphometrically and by quantitative enzyme histochemical methods (4 and 24 months only) to assess the affects of old age on the structure and activity of their neurones. DMN and NTS neuronal soma area increased whilst NA neuronal area was reduced in the aged groups; the changes in neuronal size were reflected in the density of neurones per unit area. The mean diameter and percentage area occupied by microvessels was unchanged with increased age in all three nuclei. Quantitative assessment of cytochrome oxidase and NADH-tetrazolium reductase activities in the nuclei revealed no changes in old age, indicating that old age does not affect neuronal or metabolic activity of central vagal neurones. These results compliment previous age-related studies on the vagus nerve and nodose ganglion in which little change has been reported, suggesting that the vagal system is well preserved in aged rats.

Autonomic neurons of the rat spinal cord show strong NADPH diaphorase activity and immunoreactivity for nitric oxide synthase (NOS). Here we show mRNA expression of NOS type-1 (neuronal or brain NOS) transcripts in cell bodies of sympathetic preganglionic neurons (SPNs) of the intermediolateral (IML) cell column by non-radioactive in situ hybridization using NOS-I riboprobes. Hybridization signals occurred only in neuronal cell bodies and not outside, in what appeared to be fibers and/or terminals. In preganglionic fibers of SPNs, however, dense axoplasmic immunogold labeling was detected with a monoclonal anti-NOS-I antibody. Expression of NOS-I mRNA in SPN cell bodies and axoplasmic immunolocalization of NOS-I protein suggest that protein translocation is involved in NO-mediated preganglionic control of peripheral targets.

The activity and regional distribution of D-amino acid oxidase (DAO), an enzyme that inactivates D-serine, were examined in the medulla and spinal cord of the rat by biochemical and histochemical procedures. DAO activity was noticeably low or absent in the nucleus of the solitary tract, ventrolateral medulla and intramediolateral cell column of the spinal cord. This may be indicative of a neuromodulatory role for endogenous D-serine (at the NMDA-glycine site) in in the central control of blood pressure.

Nonadrenergic-noncholinergic (NANC) inhibitory nerves are responsible for most of the nerve induced relaxations of gastrointestinal muscle. It has recently been proposed that NANC nerves may release nitric oxide (NO) or a related compound derived from L-arginine. We have recently shown that the South American (SA) opossum is another suitable model to elucidate the mechanism involved in these NANC relaxations. In the present study the effect of NO synthase inhibitors as well as NO inactivators on the NANC-nerve induced relaxations of the circular muscle of the esophagogastric junction (EGJ) of the SA opossum was investigated. It was observed that the NO synthase inhibitors, L-NOARG and L-NAME, caused a concentration-dependent reduction of NANC-nerve induced relaxations which was reversed by L- but not D-arginine. The NO-donors sodium nitroprusside and hydroxilamine as well as NO caused concentration-dependent relaxations of the EGJ circular muscle. In the myenteric plexus of this region, NADPH-diaphorase positive neurons and nerve fibers were observed while in the circular muscle layer only numerous positive fibers were found. The NO inactivators, hydroquinone, pyrogallol and carboxy-PTIO, reduced NO-induced relaxations but failed to affect NANC nerve- and sodium nitroprusside-induced relaxations. Taken together, these findings indicate that NANC nerve induced relaxation of the SA opossum EGJ circular muscle is dependent on neural NO synthase activity and suggest that the neurotransmitter being released is a superoxide resistant molecule, which is unlikely to be the NO radical, or that the activity of NO synthase is required for the release of the actual neurotransmitter rather than for synthesizing the neuromediator.

We have developed enhanced immunohistochemical protocols for detecting autonomic nerve fibers and splenocyte-associated proteins in rat spleen. This includes norepinephrine-synthesizing enzymes (dopamine-beta hydroxylase (DBH) and tyrosine hydroxylase (TH)), neuropeptide Y (NPY), tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), c-fos protein, inducible nitric oxide synthase (iNOS), and the macrophage cell marker ED1. Animals were divided into sham-operated and splenic nerve-sectioned groups for detection of DBH, TH, and NPY. For immunodetection of TNF-alpha, iNOS, IFN-gamma and c-fos, animals were injected IV with saline or 100 microg of lipopolysaccharide (LPS) and were sacrificed at various time intervals post injection. Rats were perfused with 4% paraformaldehyde, spleens removed and cryoprotected, and 50-microm floating sections were cut on a freezing microtome. Immunodetection was performed with various detection systems and substrate/chromogen solutions, and in some cases using pretreatment with proteinase K (PK) for antigen unmasking. PK pretreatment increased immunostaining for DBH, TH, NPY, IFN-gamma, iNOS, and ED1, and the improvement was concentration-dependent. Using NPY immunostaining to index the signal-to-noise ratio for various substrates and detection systems, we found that an alkaline phosphatase detection system with NBT/BCIP as a substrate was the best procedure for light microscopy, whereas the CY3-labeled secondary antibody technique proved optimal for fluorescent microscopy. Surgical transection of the splenic nerve eliminated all nerve fiber staining for DBH, TH, and NPY. TNF-alpha, IFN-gamma, c-fos, and iNOS proteins were observed in the spleen in a time-dependent manner after LPS stimulation. Fluorescent double labeling, visualized with fluorescent confocal scanning laser microscopy, revealed many NPY fibers distributed among the ED1-labeled macrophages. These results demonstrate that immunohistochemistry can be used to index the activational effects of an immune challenge on splenocytes in situ and verifies that splenic immune cells are innervated by the sympathetic nervous system.

By using mechanical nerve ligation or nerve pinch technique, we provide evidence that nitric oxide synthase (NOS) is transported in the preganglionic sympathetic axons, while postganglionic axons lack NOS transport. This finding corroborates the preganglionic sympathetic terminal as the site of NO synthesis, which is known to affect ganglionic transmission. Both vasoactive intestinal polypeptide (VIP) and substance P (SP) containing neurons of the nodose ganglion transport NOS in their axons. These results therefore suggest that NOergic innervation of autonomically innervated tissues is of parasympathetic and/or sensory, rather than sympathetic, origin.

An anatomical basis was sought for the postulated roles of nitric oxide (NO) as a labile transcellular messenger in the dorsal vagal complex (NTS-X). The diaphorase activity of NO synthase was used as a marker of neurons in NTS-X that are presumed to convert L-arginine to L-citrulline and NO. Nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) staining patterns in the nucleus tractus solitarii (NTS) were spatially related to terminal sites of primary visceral afferents from 1) orosensory receptors (e.g., rostral-central nucleus); 2) soft palate, pharynx, larynx, and tracheobronchial tree (e.g., dorsal, intermediate, and interstitial nuclei); 3) esophagus (nucleus centralis); 4) stomach (nucleus gelatinosus); 5) hepatic and coeliac nerves (nucleus subpostrema); and 6) carotid body and baroreceptors (medial commissural and dorsal-lateral nuclei). Primary visceral afferents were identified as sources of NADPHd-stained fiber plexuses in the NTS-X based on three findings: 1) the presence of NADPHd in nodose ganglion cells with morphological features of first-order sensory relay neurons; 2) retrograde transport of Fluoro-Gold (FG) or cholera toxin B (CT-B) from NTS-X to NADPHd-positive nodose ganglion neurons; and 3) striking reductions of NADPHd-stained processes within primary vagal projection fields ipsilateral to unilateral nodose ganglionectomy. A central origin of NADPHd-stained processes in NTS-X was identified in the medial parvicellular subdivision of the paraventricular hypothalamic nucleus. We conclude that NO of peripheral and central origin may modulate viscerosensory signal processing in the NTS-X and autonomic reflex function.

Synaptic connectivity and other ultrastructural features of cholinergic and non-cholinergic neurons in the laterodorsal and pedunculopontine tegmental nuclei were investigated with electron microscopy combined with pre-embedding immunohistochemistry for choline acetyltransferase. Quantitative morphometric analyses were conducted on selected immunopositive as well as immunonegative neurons. The ultrastructure of immunoreactive neurons in the laterodorsal and pedunculopontine tegmental nuclei was similar. In both nuclei, immunoreactive neurons were among the larger neurons, and somatic areas of immunopositive neurons in single thin sections were larger than those of immunonegative neurons by an average of 40%. Immunopositive somata varied in shape, appearing polygonal, fusiform or oval. Regardless of immunoreactivity, however, neurons in the pedunculopontine nucleus tended to have more irregular shapes than those in the laterodorsal tegmental nucleus. Immunoreactive neurons in both the nuclei had abundant cytoplasmic organelles and a large, clear nucleus with a few infoldings. Usually, about a quarter of the surface of an immunopositive soma was covered with astrocytic processes, and some immunopositive somata were directly apposed to an astrocyte. Immunoreactive dendrites and, less frequently, axon terminals were seen in close apposition to endothelial cells of blood capillaries or pericytes. Immunoreactive somata and dendrites in the laterodorsal and pedunculopontine tegmental nuclei received many synapses, mainly from unlabelled axon terminals. The mean number (4.7 +/- 1.8) of synapses received by immunolabelled somata in single thin sections was greater, by about 70%, than those received by unlabelled somata. The presynaptic axon terminals synapsing with immunoreactive somata commonly contained small, round and clear vesicles, and 20% of them contained a few dense-cored vesicles as well. Immunoreactive dendrites, in addition, received synapses from unlabelled axon terminals containing flat and clear vesicles, which accounted for 15% of the synapses with immunoreactive dendrites. Many immunopositive axon terminals were present in both the tegmental nuclei. They contained clear round vesicles, and usually synapsed with unlabelled dendrites. A few immunolabelled axons, however, appeared to synapse with immunopositive somata and dendrites. Immunoreactive fibres were also present in both the tegmental nuclei. They were either thinly myelinated or unmyelinated. In conclusion, the ultrastructural morphology of cholinergic neurons in the laterodorsal and pedunculopontine tegmental nuclei is similar, and these neurons represent a distinct population of neurons in both nuclei in that they are larger and receive more synaptic contacts than non-cholinergic neurons. Cholinergic neurons, however, appear to receive synapses from cholinergic axon terminals only rarely, despite the abundance of cholinergic terminals in the tegmental nuclei.(ABSTRACT TRUNCATED AT 400 WORDS)

1. We have investigated the role of phosphodiesterase isoenzymes in modulating electric field stimulation (EFS), substance P and capsaicin-induced contraction of the guinea-pig isolated main bronchus. 2. Non-adrenergic non-cholinergic contractile responses were elicited by EFS (3 Hz, 20 s) in the guinea-pig isolated main bronchus in the presence of the non-selective muscarinic antagonist, atropine (0.1 microM), the non-selective beta-adrenoceptor antagonist, propranolol (1 microM), the neutral endopeptidase inhibitor, thiorphan (10 microM) and the cyclo-oxygenase inhibitor, indomethacin (5 microM). The type III, type III/IV, type IV and type V phosphodiesterase isoenzyme inhibitor, SKF 94836, benzafentrine, Ro-20-1724 and zaprinast respectively, significantly attenuated the contractile response to EFS. The IC50 (95% confidence limits) value for SKF 94836, benzafentrine, Ro-20-1724 and zaprinast was 8.3 microM (0.89-78); 0.7 microM (0.1-4.5); 0.5 microM (0.2-1.2) and 13 microM (2-87) respectively. 3. The phosphodiesterase isoenzyme inhibitors, SKF 94836, Ro-20-1724 and zaprinast, partially attenuated the contractile response to substance P (10 nM). Benzafentrine significantly inhibited the contractile response to substance P, yielding an IC50 value of 1.9 microM (0.9-3.8). 4. The phosphodiesterase isoenzyme inhibitor, Ro-20-1724 (0.1-100 microM) failed to reduce significantly the contractile potency of capsaicin (P > 0.05). In contrast, SKF 94836 (1 microM), benzafentrine (10 microM) and zaprinast (100 microM) significantly reduced the contractile potency of capsaicin (P < 0.05). 5 The selective phosphodiesterase isoenzyme inhibitors, SKF 94836, benzafentrine, Ro-20-1724 andzaprinast (0.01-100 microM) reversed in a concentration-dependent manner the contractile response toexogenously administered capsaicin (EC50) yielding ICm values of 3.91 microM (0.68-22); 3.37 microM (1.86-6.11); 0.366 microM (0.201-0.564) and 50.1 microM (18.6- 135) respectively.6 In conclusion, phosphodiesterase isoenzymes appear to regulate the contractile response to electricalfield stimulation and our results provide circumstantial evidence for a regulatory role ofphosphodiesterase type IV isoenzyme on sensory nerve function in vitro.

In the cat carotid body, nitric oxide synthase (NOS) immunoreactivity and NADPH diaphorase activity localize in nerve fibers mainly associated with blood vessels and occasionally lying close to glomus cells. The NOS-positive innervation originates in part from multipolar ganglion cells scattered in and around the carotid body and in the glossopharyngeal nerve. In the superior cervical ganglion, NOS and diaphorase staining localizes to many preganglionic axons and also to a small population of vasoactive intestinal peptide-positive, presumably cholinergic, ganglion cells. Positively stained ganglion cells are absent in the petrosal ganglion and very rare in the nodose ganglion, although both sensory ganglia display characteristic distributions of cells immunoreactive for calcitonin gene-related peptide, substance P and tyrosine hydroxylase. The NOS-positive innervation of the carotid body thus appears to be autonomic, originating mainly from a population of dispersed ganglion cells, and probably parasympathetic in nature. The superior cervical ganglion also may supply some pre- or postganglionic NOS-positive axons. Nitric oxide released from these nerves could affect glomus cell activity directly or indirectly by vasoregulation.

A previous in situ hybridization study by Wisden et al., J. Neurosci. 12, 1040-1062, showed a high expression of the GABAA receptor alpha 1, beta 2 and gamma 2 subunit messenger RNAs in the medial septal nucleus and nucleus of the diagonal band of the rat. The aim of the present study was to determine whether this high expression of GABAA receptor subunit messenger RNAs is found in the cholinergic neurons of these areas. Adjacent 4-5 microns sections of rat forebrain were submitted to choline acetyltransferase immunocytochemistry or to in situ hybridization histochemistry using oligonucleotides complementary to parts of the GABAA receptor alpha 1, alpha 2, beta 2, delta 1 and gamma 2 subunit messenger RNAs. It was found that the high expression for the GABAA receptor alpha 1, beta 2 and gamma 2 subunit messenger RNAs in the medial septal nucleus, the nucleus of the diagonal band and the nucleus basalis is located almost exclusively in non-cholinergic neurons. It was also found that these non-cholinergic cells are also continuous in distribution with neurons in the globus pallidus and ventral pallidum that similarly express high levels of messenger RNA for the GABAA receptor alpha 1, beta 2 and gamma 2 subunits. It was concluded that the basal forebrain cholinergic neurons may not be as sensitive to GABAA receptor influences as their non-cholinergic neighbours.

The liver innervation of eight different mammalian species was examined by immunohistochemical localization of protein gene product (PGP) 9.5 to visualize the general innervation for autonomic nerve fibres. In addition, dopamine beta-hydroxylase (DBH) and tyrosine hydroxylase (TH), two enzymes involved in catecholamine synthesis, were localized immunohistochemically to delineate hepatic sympathetic nerve fibres. We found that: (1) Within the interlobular region of each species, PGP 9.5, DBH and TH-positive nerve fibres were all seen in close association with branches of hepatic arteries, portal veins and bile ducts. (2) Within the parenchyma of the guinea-pig, cat, dog, pig, monkey and human liver, the presence of the three immuno-positive nerve fibres could be unequivocally identified, although the density of these intralobular fibres showed marked species variation. Moreover, immunoelectron microscopic study confirmed that PGP 9.5-positive nerve terminals of the human liver are in close apposition to hepatocytes. (3) In mouse and rat, no parenchymal nerve fibres immunoreactive for PGP 9.5, TH or DBH could be demonstrated.

Purpose: To determine if nitric oxide (NO) is involved in the relaxant response to nerve stimulation by nicotine and electrical pulses in dog external ophthalmic arteries (EOA) and internal ophthalmic arteries (IOA)., Methods: Changes in isometric tension were recorded in helical strips of the arteries, and the presence of perivascular nerve containing NADPH diaphorase was histochemically demonstrated., Results: Nicotine (10(-4) M, EOA and IOA) and transmural electrical stimulation (5 Hz, EOA) produced a slight or no contraction followed by a moderate relaxation in the strips contracted with prostaglandin F2 alpha. The contraction was abolished by alpha-adrenoceptor antagonists. The relaxation was abolished and the contraction was potentiated by NG-nitro-L-arginine (L-NA), a nitric oxide (NO) synthase inhibitor; the relaxation was reversed by L-arginine. Contractile response in L-NA-treated EOA was greater than in the IOA, and the relaxation was less in nontreated EOA. NO-induced relaxation and norepinephrine-induced contraction were not influenced by L-NA. There were plenty of nerve fibers visualized by NADPH diaphorase staining method in the adventitia of EOA and IOA, indicating the presence of NO synthase-containing nerves., Conclusions: The neurogenic relaxation appears to be mediated by NO released from the vasodilator nerve in EOA and IOA. There is a reciprocal innervation in vasodilator nitroxidergic and vasoconstrictor noradrenergic nerves; functionally, the latter is more predominant in EOA than in IOA.

Recently, we observed that atrial natriuretic peptide (ANP) immunoreactivity (IR) was present not only in the Purkinje fibres, but also in nerve fibre varicosities in the conduction system of the bovine heart. These findings and previous observations that ANP is able to influence autonomic neurotransmission in the heart, lead us to elucidate the possible occurrence of ANP in the sympathetic and/or parasympathetic nervous systems and/or in various types of peptidergic innervation in the conduction system. The different parts of the conduction system of bovine hearts were dissected out and processed for immunohistochemistry including double-staining, using antisera against ANP, tyrosine hydroxylase and different neuropeptides. We observed that some of the nerve fibre varicosities exhibiting ANP-IR showed substance P-IR and that ANP was present as scattered immunoreactive granules in intracardial, presumably parasympathetic, ganglionic cells. The study shows that ANP is likely to be present in parasympathetic innervation and in afferent nerve endings in the bovine heart conduction system.

In the present immunohistochemical study the distribution of nitric oxide synthase (NOS) was studied in various autonomic ganglia and in related peripheral tissues of the rat. For comparison some other neuronal markers including acetylcholinesterase and tyrosine hydroxylase as well as several neuropeptides were analysed on adjacent or the same sections. The distribution of NOS-like immunoreactivity (LI) and of these other markers has been semiquantitatively summarized. In some parasympathetic ganglia such as the sphenopalatine and submandibular ganglia NOS-LI was present in most ganglion cells, at least partly coexisting with peptide histidine isoleucine (PHI), vasoactive intestinal polypeptide (VIP) and neuropeptide tyrosine (NPY). In the pelvic ganglia a comparatively smaller proportion of neurons was NOS-positive and they often contained VIP-LI and less frequently NPY-LI. In the tissues innervated by these ganglia, such as nasal mucosa and salivary glands, NOS-positive fibers were observed around blood vessels and within the glandular parenchyma, although generally less abundant than VIP/PHI nerves, while in the uterus, vas deferens and penis a more close correlation was seen. NOS-positive fibers were also widely distributed in other tissues. In the sympathetic ganglia NOS-LI was mainly present in dense fiber networks, which disappeared after transection of the sympathetic trunc central to the ganglion. Since many cell bodies in the sympathetic lateral column of the spinal cord also were NOS-positive, it is likely that the majority of preganglionic fibers innervating sympathetic ganglia are NOS-positive. VIP-positive cells in stellate ganglia did not contain NOS-LI. The present results suggest that NO may be a messenger molecule both in parasympathetic postganglionic neurons and in preganglionic sympathetic neurons.

Nitric oxide synthase, the enzyme responsible for the formation of nitric oxide, was demonstrated by an indirect immunofluorescence technique to be present in both the sympathetic and parasympathetic nervous system of the domestic pig. In the sympathetic nervous system, nitric oxide synthase was mainly present in preganglionic neurons projecting to postganglionic neurons, some of which contained neuropeptide Y in the superior cervical, the coeliac and the lumbar ganglia of the sympathetic chain. A minor population of postganglionic sympathetic neurons contained nitric oxide synthase, vasoactive intestinal polypeptide and peptide histidine isoleucine. In the densely sympathetically innervated vascular beds such as the spleen, kidney and skeletal muscle, many neuropeptide Y- but no nitric oxide synthase-positive fibres were found. The nitric oxide synthase inhibitor NG-nitro-L-arginine reduced cardiac output by 40% and caused profound vasoconstriction in a variety of vascular beds. Furthermore, no or minor changes in plasma catecholamines, neuropeptide Y or endothelin-1 were observed up to 20 min after NG-nitro-L-arginine. Milrinone (a phosphodiesterase III inhibitor) prevented this NG-nitro-L-arginine-induced reduction in cardiac output, and the regional vasoconstriction was reduced, whereas some elevation of the blood pressure was still observed. Sympathetic nerve stimulation, with single impulses of 10 Hz for 1 s in the presence of NG-nitro-L-arginine, evoked vasoconstrictor responses which were largely in the same range as in control conditions. Parasympathetic postganglionic neurons to the submandibular salivary gland contained nitric oxide synthase, vasoactive intestinal polypeptide, peptide histidine isoleucine and neuropeptide Y. The vasodilatation evoked by parasympathetic nerve stimulation (10 Hz for 1 s) in the presence as well as in the absence of atropine was, on the other hand, markedly reduced by NG-nitro-L-arginine administration. Milrinone attenuated the inhibitory effect of NG-nitro-L-arginine on the parasympathetic vasodilation. In conclusion, nitric oxide synthase can be demonstrated in preganglionic sympathetic and postganglionic parasympathetic neurons. The main effect of nitric oxide synthase inhibition seems to be related to attenuation of basal endothelial nitric oxide production and parasympathetic transmission. Inhibition of phosphodiesterase counteracts both the haemodynamic and the neuronal effects of NG-nitro-L-arginine.

A subpopulation of interstitial cells (ICs) are interposed between nerve terminals and smooth muscle cells in the gastrointestinal tract and may participate in neuromuscular transmission. These cells appear to be targets for NO released from enteric inhibitory nerves and respond to exogenous NO with: (i) an elevation in cGMP levels; (ii) an increase in intracellular Ca2+; (iii) and release of a diffusible substance that has tentatively been identified as NO. For the latter to be possible, ICs must express a constitutive isoform of NOS. This study characterized the expression of NOS-like immunoreactivity (NOS-LI) in ICs of the canine colon using 3 antibodies raised against the 2 known constitutive forms of NOS (i.e., neural (nNOS) and endothelial (eNOS) isoforms). Antibodies raised against cNOS and an antibody raised against rat cerebellar nNOS labeled ICs along the submucosal surface of the circular muscle layer (IC-SM), along the surface of septa that separate the circular muscle into fiber bundles (IC-SM), and in the myenteric region between the circular and longitudinal muscle layers (IC-MY). Another antibody raised against rat cerebellar nNOS failed to label ICs. Cultured IC-SM also expressed NOS-LI, suggesting that this feature of the IC phenotype survives culture conditions. Arteriolar endothelial cells in the canine colon were labeled with the same 2 antibodies that labeled ICs, suggesting there are significant structural similarities between NO synthases in ICs and endothelial cells. The data suggest that IC-SM and IC-MY express a constitutive form of NOS. Synthesis of NO by ICs may influence electrical rhythmicity and may serve to amplify and even propagate enteric inhibitory neurotransmission.

Neuronal origins, peptide phenotypes and target distributions were determined for sensory and autonomic nerves projecting to the eyelid. The retrograde tracer, Fluoro-Ruby, was injected into the superior tarsal muscle and meibomian gland of Sprague-Dawley rats. Labelled neurons were observed within the pterygopalatine (31 +/- 6 of a total of 8238 +/- 1610 ganglion neurons), trigeminal (173 +/- 43 of 62,082 +/- 5869) and superior cervical ganglia (184 +/- 35 of 21,900 +/- 1741). Immunostaining revealed vasoactive intestinal polypeptide immunoreactivity (VIP-ir) in nearly all Fluoro-Ruby-labelled pterygopalatine ganglion neurons (86 +/- 5%) but only rarely in trigeminal (0.3 +/- 0.3%) or superior cervical (1.4 +/- 1.4%) ganglion neurons. Calcitonin gene-related peptide (CGRP)-ir was not observed in pterygopalatine or superior cervical ganglion somata, but was present in 24 +/- 4% of trigeminal neurons. Bright dopamine beta-hydroxylase (DBH) immunofluorescence was observed in the majority of eyelid-projecting neurons within the superior cervical ganglia (65 +/- 5%) and lighter staining was detected in pterygopalatine neurons (63 +/- 3%), but no DBH-ir was observed in trigeminal neurons. Examination of eyelid sections revealed dense VIP-ir innervation of meibomian gland acini and vasculature and modest distribution within tarsal muscle. CGRP-ir fibers surrounded ductal and vascular elements of the meibomian gland and the perimeter of tarsal muscle. DBH-ir fibers were associated with meibomian gland blood vessels and acini, and were more densely distributed within tarsal muscle. This study provides evidence for prominent meibomian gland innervation by parasympathetic pterygopalatine ganglion VIP-ir neurons, with more restricted innervation by sensory trigeminal CGRP-ir and sympathetic neurons. Tarsal muscle receives abundant sympathetic innervation, as well as moderate parasympathetic and sensory CGRP-ir projections. The eyelid contains substantial non-CGRP-ir sensory innervation, the targets of which remain undetermined. The distribution of identified autonomic and sensory fibers is consistent with the idea that meibomian gland function, as well as that of the tarsal muscle, is regulated by peripheral innervation.

We have used a polyclonal monospecific antibody to rat cerebellum nitric oxide synthase type I (NOS-I, 160 kD) in combination with reduced NADPH-diaphorase histochemical reaction (NADPH-d) to verify colocalization of both NOS protein and NOS enzymatic activity in the rat spinal cord autonomic system. Strong immunoreactivity (IR) of NOS-I was clearly detected in the four main thoracolumbar autonomic nuclei in spinal cord layers of Rexed's laminae VI, VII and X. In all labelled neurones, NOS-I-IR colocalized with NADPH-d activity, suggesting coexistence of brain-specific NOS-I-like protein and its associated enzyme activity. For these neurones the new term 'nitroxergic' (i.e. NO-generating) neurones appears to be justified. Spinal cord nitroxergic neurones are part of a NO-mediated signal transduction pathway for control of the sympathetic 'outflow' to various peripheral target organs.

Expression of the trkA receptor for nerve growth factor was analyzed by mRNA in situ hybridization in the telencephalon and diencephalon in adult rats. Two densely packed populations expressing trkA mRNA were detected in the thalamus, in the paraventricular thalamic nucleus anterior and reuniens thalamic nucleus. These neurons did not express mRNA coding for choline acetyltransferase. In basal forebrain and striatum, trkA and choline acetyltransferase mRNAs showed identical patterns of expression, supporting the view that trkA expression is confined to cholinergic population in these areas. In contrast, cholinergic neurons of the medial habenula did not express trkA mRNA. Our findings suggest the existence of previously undiscovered populations of non-cholinergic, NGF responsive populations in the thalamis.

The synaptosomes from canine ileal deep muscular plexus possess a nitric oxide (NO)-sensitive soluble guanylate cyclase, as demonstrated by approximately 3- to 4-fold elevation of synaptosomal cyclic GMP levels in the presence of either 1 mM sodium nitroprusside or L-arginine (20-1,000 microM) plus 1 mM NADPH. The activating effect of L-arginine on synaptosomal soluble guanylate cyclase was related to its enzymatic conversion to citrulline by NO synthase. The synaptosomal NO synthase was found to exhibit both calcium-independent and calcium/calmodulin-dependent components accounting for approximately 2- to 2.5-fold and 7- to 8-fold increases in the basal activity, respectively. The absolute magnitude of these activities was several-fold greater compared to the activities observed in the isolated cells of circular smooth muscle. The synaptosomal Ca-independent and Ca/calmodulin-dependent NO synthase activities were inhibited by methylene blue and L-NG-arginine methyl ester. The NO synthase activity was also attenuated in the presence of cyclic AMP (10 microM). Such an inhibition was related primarily to the suppression of Ca-independent activity. The ability of enteric nerves to generate NO from L-arginine strongly suggests the involvement of this process in the biochemical mechanisms underlying the neurogenic control of intestinal motility.